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Introduction

The BioModule™ Immunohistochemical (IHC) Staining for Tissue provides qualified reagents and validated protocols to perform highly sensitive and specific immunohistochemical staining of tissues. The key component of Bio-Module™ IHC Staining Unit for Tissues is the SuperPicTure™ Polymer Detection Kit employing HRP detection system to detect mouse, rabbit, rat, or guinea pig primary antibodies bound to tissue antigens. The BioModule™ IHC Staining for Tissues is designed for use with frozen and formalin-fixed, paraffin-embedded tissues.

In addition to the immunohistochemical staining reagents, the BioModule™ IHC Staining Unit for Tissues also includes several key reagents for peroxidase quenching, counterstaining, and mounting.

BioModule™ Units for Gene Expression Profiling

The Bio-Module™ IHC Staining Unit for Tissues is one of the several BioModule™ Units available from Invitrogen for gene expression profiling. Each of the BioModule™ Units for gene expression profiling includes high-quality reagents and validated protocols with relevant controls for each step of the workflow (see below). Each unit is designed to provide an integrated workflow that allows you to perform various steps seamlessly during expression analysis. Gene expression profiling comprises multiple steps employing various technologies such as microarray analysis or quantitative PCR (qPCR) for analysis at the nucleic acid level; western immunodetection and immunohistochemistry for analysis at the protein level; and RNAi for functional analysis.

IHC

Immunohistochemical (IHC) Staining allows you to detect antigens in a tissue fixed on a glass slide. The BioModule™ IHC Staining Unit utilizes indirect staining method to detect antigens and includes the following steps:

Enzyme (HRP) forms a colored (brown), insoluble precipitate at the antigenic sites in the presence of a substrate (H2O2) using a chromogen, DAB (3, 3'-diaminobenzidine) that is easily visualized with light microscopy. IHC staining is simple and easy to perform at the benchtop without the need for any specific instrumentation and allows you to detect antigens in context of tissue morphology. The colored precipitate from DAB is stable for several years providing a permanent record.

Materials

Introduction

Brief description of the components included with the BioModule™ IHC Staining Unit for Tissues is described in this section.

SuperPicTure™ Kit

SuperPicTure™ Kit is one-step polymer detection kit using HRP and DAB for increased sensitivity. The kit employs a stable amino acid polymer conjugate containing multiple HRP’s that react with mouse, rabbit, guinea pig, and rat primary antibodies. Since the SuperPicTure™ Kit does not contain biotin or streptavidin, there is no background due to endogenous biotin activity. The SuperPicTure™ Kit is designed for use with frozen or formalin-fixed paraffin embedded tissues and user supplied primary antibodies. The SuperPicTure™ Kit contains the HRP Polymer Conjugate, 20X DAB substrate solution, 20X buffer for diluting the substrate, and 20X 0.6% H2O2.

Peroxo-Block™

Peroxo-Block™ is a ready-to-use solution containing specific inhibitor of endogenous peroxidase activity. The Peroxo-Block™ is specially formulated for frozen or formalin-fixed paraffin embedded tissues and effectively eliminates endogenous peroxidase activity in less than a minute without interfering with specific immunostaining.

Hematoxylin Counterstain Reagent

Hematoxylin Counterstain Reagent is a ready-to-use, bluish/purple counterstain for routine nuclear staining and can be used with many chromogens including DAB. Hematoxylin Counterstain Reagent stains nuclei brilliantly without interfering with the chromogen signal. The reagent produces a brilliant blue nuclear counterstain for immunohistochemistry.

Histomount™

Histomount™ is a ready-to-use organic based mounting medium for immunohistochemical procedures and ideal for preserving organic insoluble chromogens such as DAB. Samples preserved in Histomount™ are stable for several years.

Mini PAP Pen

The Mini PAP Pen allows you to draw a water repellent circle around the tissue mounted on a slide, keeping the liquid pooled in a single droplet. This prevents any wastage of valuable reagents and ensures even staining of the tissue. The Mini PAP Pen is especially useful when working with multiple sections on a single slide. Each pen can be used to draw about 400 circles.

PBS and Antibody Diluent

PBS

Use the PBS powder included with the kit to prepare 1X PBS (10 mM phosphate buffer, pH 7.2-7.3 with 150 mM NaCl). The PBS with 0.05% Tween-20 is used for some washing steps during the staining protocol.

The Antibody Diluent is a ready-to-use solution of 1X PBS with BSA (for stabilizing the antibody) and preservative. Dilute your primary antibody using the Antibody Diluent, if you are not using a pre-diluted antibody such as the Zymed® 2nd Gen antibodies.

General Guidelines

Introduction

General guidelines for using the BioModule™ IHC Staining Unit for Tissues are described in this section. Review the information in this section prior to performing immunohistochemical staining to obtain the best results.

Most of the reagents included with the BioModule™ IHC Staining Unit for Tissues are supplied as ready-to-use solutions without the need for any preparation or dilution and are supplied in a dropper bottle that allows easy dispensing of reagents. We do not recommend diluting the reagents beyond the concentration supplied or removing the reagents from dropper bottles. Do not pipette reagents directly from the bottle.

If you are performing immunohistochemical staining using mouse or rat primary antibodies on mouse or rat tissues, respectively, we recommend that you use HistoMouse™-Max Kit to prevent background problems and obtain best results.

Starting Material

The BioModule™ IHC Staining Unit for Tissues is designed for use with frozen or formalin-fixed paraffin embedded tissue sections mounted on slides using primary antibodies.

Based on your starting sample material, you may need to perform some sample preparation steps, prior to staining the tissue sections.
Fixatives

Cell smears prepared from body fluids should be made to assure a monolayer of cells. Multilayers of cells can trap staining reagents and interfere with the interpretation of results. Fix smears immediately after preparation. Depending on the properties of the antigen, cell smears are usually stable for 1-2 weeks when stored at 4°C.

Primary Antibody

The BioModule™ IHC Staining Unit for Tissues is compatible for use with any mouse, rabbit, guinea pig, and rat primary antibodies. A large variety of high-quality antibodies including the Zymed® Antibodies is available from Invitrogen for use in immunohistochemistry. Prediluted 2nd Gen primary antibodies that are ready-to-use and titered for use with the SuperPicTure™ Polymer Detection Kit are also available. 2nd Gen primary antibodies contain a general protein blocker eliminating the need for a separate blocking step.

Polyclonal or monoclonal antibodies can be used with the kit. For higher specificity, we recommend using monoclonal antibodies. Select antibodies that can detect low antigen levels and can recognize epitopes from formalin-fixed paraffin embedded tissues.

The optimal antibody concentration for use with immunohistochemistry is usually recommended by the antibody manufacturer or you may determine the optimal concentration using a checkerboard titration experiment.

Because different tissues have different levels of endogenous peroxidase activity, you may need to perform the peroxidase quenching step, only if your tissue exhibits high endogenous peroxidase activity.

To assess the endogenous peroxidase activity in your tissue, hydrate the slide containing tissue sections in PBS. Apply the DAB substrate, incubate for 10 minutes, and wash with distilled water. Examine the slide under the microscope to see any staining. If there is staining, you need to perform the peroxidase quenching step using Peroxo-Block™.

Epitope Retrieval

Antigens that are masked by formalin fixation and embedding procedures can be retrieved using standard proteolytic or heat treatment procedures prior to performing the immunohistochemical staining. Some antibodies require epitope retrieval as recommended by the antibody manufacturer while staining with some antibodies is enhanced by epitope retrieval.

Appropriate Controls

When performing immunohistochemical staining, it is important to include proper positive and negative controls to help evaluate your results.

We recommend including the following three control slides that are necessary for interpreting results. Additional controls can be added based on the experimental design.

Positive Tissue Control

A specimen processed in the same way as the unknown and contains the antigen to be stained.

Reagent Control

An additional slide that is treated with a non-immune serum or isotype control antibody that matches the isotype of the experimental antibody instead of the same concentration of primary antibody. Any staining observed on the specimen is probably due to non-specific protein binding or non-specific binding of other reagents.

Rabbit and mouse primary antibody isotype controls are available from Invitrogen.

Negative Control

A specimen processed in the same way as the unknown but does not contain the antigen to be stained (optional).

Sample Preparation

Introduction

This section provides general guidelines for sample preparation to perform immunohistochemical staining. Proper sample preparation is key to the success of an immunohistochemical staining experiment. Based on your starting sample material, you may need to perform some sample preparation steps described below, prior to staining the sample.

Detailed protocols for preparing frozen or formalin-fixed paraffin embedded tissues or tissue sectioning are not included in this manual.

Materials Needed

You will need the following materials:

Fresh, frozen tissue, or paraffin embedded tissue of choice

Microtome and cryostat for tissue sectioning

Pre-coated slides (see below)

Coplin jars or equivalent

PBS (supplied with the kit)

For Frozen sections

OCT® Compound

Chilled 100% acetone for fixing

For formalin-fixed paraffin embedded sections

Xylene

Graded series of ethanol (80%, 95% and 100% ethanol)

55ºC oven

Slide Preparation

If you are preparing your own slides, pre-coat slides with HistoGrip™ or 0.1% poly-L-lysine in water, then air dry. Commercially available pre-coated glass slides are available and can be used to mount frozen or formalin-fixed paraffin embedded tissue sections.

Frozen Tissue

A sample protocol for preparing frozen tissue samples is described below. If you have optimized protocols in the laboratory for your sample type, use the optimized protocol.

For sectioning, allow the frozen tissue block to equilibrate to the cryostat temperature.

Cut 4-6 µm cryostat sections and mount on coated glass slides.

Dry tissue sections at room temperature for 30 minutes. If desired, store slides at -70ºC before fixing. If slides are stored at -70ºC, warm the slides to room temperature before the fixing step.

Place the slides in 100% acetone at 4ºC for 10 minutes to fix the sections.

Remove slides from acetone and air dry for 10-30 minutes.

Circle each tissue section using the Mini PAP Pen.

Store at -70ºC until use or wash the slide in PBS for 10 minutes and proceed immediately to Peroxidase Quenching.

Paraffin Embedded Sections--Deparaffinization and Rehydration

To use the formalin-fixed paraffin embedded sections for immunohistochemical staining, you need to perform the deparaffinization with xylene and rehydration in a graded series of alcohol as described in the sample protocol below.

Obtain or prepare the formalin-fixed paraffin embedded sections of choice.

Proceed immediately to Incubating with Primary Antibody, or proceed to Epitope Retrieval Protocol

Peroxo-Block™ works efficiently on many tissues, especially with formalin-fixed paraffin embedded tissues, but with some frozen tissues, you may need to use 3% H2O2 to obtain optimal results (better morphology or lower background). To use 3% H2O2 for blocking endogenous peroxidase activity, incubate the slides in 3% H2O2 in methanol for 10 minutes. Wash slides 3 times with 1X PBS for 2 minutes, each time and then proceed to primary antibody incubation.

Epitope Retrieval Protocol

A sample epitope retrieval protocol developed for some Zymed® Antibodies is described below. The epitope retrieval protocol is used to reverse the loss of antigenicity that occurs with some epitopes in formalin-fixed paraffin embedded tissues. Some Zymed® antibodies may require epitope retrieval protocol (e.g., estrogen receptor, and Ki-67 antibodies), while the staining of many other antibodies may be enhanced by epitope retrieval protocol (e.g., S-100, cytokeratin, and synaptophysin antibodies).

You may use any other epitope retrieval protocol recommended by the supplier of your primary antibody.

Heat Induced Epitope Retrieval Protocol

Perform this epitope retrieval protocol after Peroxidase Quenching. Note: You may use citrate buffer or EDTA solution depending on the antibody manufacturer’s recommendation.

Based on the reagent that you are using, dilute the reagent to 1X as follows:

Place the slides in a slide rack and place the rack in a 1 L glass beaker containing 500 ml diluted citrate buffer (1X), pH 6.0 or diluted EDTA (1X) solution, pH 8.0.

Place the beaker with slides on a hot plate. Heat the solution until it boils and continue boiling the solution for 15 minutes.

Remove beaker from the hot plate and allow the contents to cool for 25 minutes at room temperature.

Rinse slides with 1X PBS and proceed immediately to Incubation with Primary Antibody

Epitope Retrieval Protocol Using Enzymatic Digestion

Brief protocol using Digest-All™ Kit is described below. The Digest-All™ is a flexible and standardized system for tissue digestion and contains trypsin, ficin, and pepsin proteolytic enzymes. The degree of digestion is based on a standardized 10 minute incubation at 37°C. The trypsin solution is supplied with diluent so that different trypsin concentrations can be made to generate a range of digestion activity between that of Ficin and Pepsin.

Perform this epitope retrieval protocol after Peroxidase Quenching

Add the digestion enzyme of choice to your tissue sections. Ficin and pepsin may be applied directly from the bottle. Trypsin requires dilution: 1 drop Trypsin concentrate to 3 drops Trypsin Diluent. Mix well and apply to tissue sections.

Incubate for 10 minutes at 37°C.

Wash slides in several changes of 1X PBS and proceed immediately to Incubation with Primary Antibody.

Incubation with Primary Antibody

For best results, primary antibody dilution and incubation times need to be determined empirically and are dependent on sample preparation, antibody affinity, amount of antigen present, and antigen accessibility. The HRP Polymer Conjugate will react with mouse, rabbit, guinea pig, and rat primary antibodies.

Dilute primary antibody in Antibody Diluent as recommended by the antibody manufacturer or as determined by a titration experiment. Alternatively, you may use prediluted 2nd Gen primary antibodies.

Apply 100 µl or enough of the diluted primary antibody to completely cover the tissue on each section.

Troubleshooting

Review the information in this section to troubleshoot your immunohistochemical staining experiments.

Problem

Reason

Solution

Weak or no staining

Primary antibody was inactive or too dilute

Perform staining using a new batch of antibodies.

Be sure to store the antibody at the recommended temperature. Aliquot the antibody into smaller aliquots and store as recommended avoiding repeated freezing and thawing that may result in loss of activity. Check the activity of the antibody using ELISA or Western blotting.

Be sure to perform a titration experiment to determine the optimal antibody concentration.

Insufficient incubation times

Be sure to perform all incubation steps as indicated in the protocol. You may increase the antibody incubation time to improve staining or increase the substrate incubation time.

Missed steps or steps not performed in the correct order

Make sure the staining protocol was followed

Incomplete deparaffinization

Perform the deparaffinization step for a longer time. Use fresh xylene.

Do not allow the sections to dry out during the staining protocol. Be sure to add enough reagents to completely cover the sections on the slide (usually 100-200 µl). Perform all incubations in a covered humidified chamber to prevent drying.

Avoid trapping any bubbles when placing the cover slip on the slide. If bubbles are already trapped, remove the cover slip by incubating the slide in a 37ºC water bath until the cover slip can be removed easily. Place a clean cover slip on the slide without trapping any bubbles.

Negative staining on positive slides

Specimen was improperly fixed or processed

Refer above the recommended sample preparation protocols.

Missed primary antibody/HRP Polymer Conjugate incubation steps or steps not performed in the correct order

Make sure the staining protocol was followed.

Sections on the slide have dried

Do not allow the sections to dry out during the staining protocol. Be sure to add enough reagents to completely cover the sections on the slide (usually 100-200 µl). Perform all incubations in a covered humidified chamber to prevent drying.

Weak or no staining for the antigen in question but there is a precipitate on the slide

Primary antibody contaminated

Use fresh batch of primary antibody. Use sterile pipette tips while handling reagents to prevent contaminating the antibody solution.